The research concerns naturally occurring protein inhibitors of proteolytic enzymes, in particular, their molecular mechanisms of action and the development of methods for their study in biological systems as well as their roles in biological systems. Particular inhibitors studied will be avian ovomucoids, avian ovoinhibitors, bovine and human pancreatic inhibitors and bovine and human plasma inhibitors (in particular, the alpha-1-trypsin inhibitor). The enzymes studied will include bovine alpha-chymo-trypsin and trypsin, human fractions of chymotrypsin and trypsin, bacterial subtilisin, plant proteinase (ficin), and various other proteinases, such as kallikreins, of animal (including human) origin. The main approaches will include studies on the relative roles of the non-covalent interactions and covalent interactions (resulting from catalytic attack of the enzyme on the inhibitor) in the inhibitory mechanism. These will be accomplished by studying the interactions under different physical and chemical environments and by studying the direct role of catalytic activity through studies with catalytically inert enzymes (such as anhydroserine and N-methylhistidine derivatives of trypsin and chymotrypsin) and examinations of the importance of resistance of the inhibitor to proteolysis. Application of these studies will be made to the determination, isolation and function of inhibitors in biological systems. In particular, catalytically inert enzymes will be used for these purposes.